A large portion of tartrate resistant acid phosphatase (TRACP) in serum is considered acid phosphatase derived from osteoclast and the assay of TRACP is considered useful as an indication for evaluating the function of osteoclast. Thus, TRACP is gaining interest as a bone resorption marker (Norio Fukunaga, Toshitaka Nakamura and Toshio Matsumoto, “Osseous Metabolism Marker”, Medical Review Co., Ltd., 1995). On the other hand, acid phosphatase in serum is divided into six bands 0 to 5 from the origin by polyacrylamide gel electrophoresis. Of these, acid phosphatase corresponding to the fifth band is tartrate-resistant and is called Band 5 tartrate resistant acid phosphatase (TRACP 5: tartrate resistant acid phosphatase 5). This acid phosphatase is further divided by electrophoresis into 5a which has a high content of sialic acid bonded to a sugar chain and 5b which has almost no sialic acid bonded to a sugar chain. In addition, 5a is an enzyme derived from cells other than osteoclast and its blood level does not vary, while only the blood level of 5b varies with bone resorption. Therefore, it is considered that 5b is the main substance of tartrate resistant acid phosphatase derived from osteoclast. Also in “Clinical Chemistry” (Clin. Chem. 47: 1497. 2001), it is recommended that ACP derived from osteoclast should be abbreviated as TRACP 5b. Accordingly, also in the present specification, phosphatase that refers to ACP derived from osteoclast and used as an indication of bone resorption is expressed in the term “TRACP 5b”, and tartrate resistant acid phosphatase derived from osteoclast and tartrate resistant acid phosphatase 5b are hereinafter considered synonymous with each other. Thus, all of them are hereinafter expressed in the term “TRACP 5b” in the present specification.
Conventional activity assay methods in which TRACP activity is assayed as an indication of acid phosphatase capable of indicating the activity of osteoclast are disadvantageous in specificity, sensitivity, troublesomeness of assay and assay time.
In general, in the assay of TRACP 5b by the activity assay method, the enzymatic activity is assayed by colorimetric determination of a reaction product (an alcohol or a phenol) produced by an enzyme reaction, by using a phosphoric ester as a synthetic substrate in the presence of tartaric acid. In this case, tartaric acid inhibits acid phosphatase derived from prostate and the residual acid phosphatase activity is assayed with the substrate to assay TRACP activity as TRACP 5b activity. However, besides tartrate resistant acid phosphatase derived from osteoclast, that derived from erythrocyte or that derived from platelet is present in a specimen, and such acid phosphatase is also assayed. Therefore, the above TRACP 5b assay method is disadvantageous in specificity.
As a modification of such a method, there is known a method in which after a pretreatment comprising incubation of a 5-fold dilution of serum at 37° C. for 1 hour, the residual TRACP activity is assayed by the use of p-nitrophenylphosphoric acid as a substrate in the presence of tartaric acid (“Nichidai-Ishi”, 49:904-911. 1990; and Clin. Chem. 33:458-462. 1987). This method permits avoidance of the influence of acid phosphatase derived from erythrocyte but does not permit exclusion of the influence of acid phosphatase derived from platelet. In addition, the present inventors have reported a TRACP 5b assay method utilizing the difference in sensitivity to fluorine between TRACP 5b and tartrate resistant acid phosphatase activity derived from erythrocyte or platelet, as a more specific activity assay method (JP-A-10-37198). This method, however, does not permit exclusion of the influence of TRACP 5a though it permits avoidance of the influence of tartrate resistant acid phosphatase derived from erythrocyte or platelet. Moreover, this method is disadvantageous in precision because TRACP 5b activity is assayed in this method by subtracting activity not inhibited in the presence of fluorine from the total tartrate resistant acid phosphatase activity. In addition, a method has been reported in which TRACP 5b activity is assayed by using an inhibitor for TRACP 5a in combination with the above-mentioned method utilizing fluorine (JP-A-2001-231595). However, since TRACP 5b activity is assayed by the subtraction also in this method, this method is disadvantageous in precision like the method using only fluorine, though it is more specific than the latter method.
On the other hand, as TRACP 5b assay methods using immunoassay, there are also known immunoassay methods using a polyclonal antibody or a monoclonal antibody (J Clin Endocrinol Metab. 71:442-451. 1990; J Bone Miner Res. 13:683-687. 1998; Immunol Lett. 70:143-149. 1999; J Bone Miner Res. 14:464-469. 1999; Clin Chem. 45:2150-2157. 1999; and Clin Chem. 46:1751-1754. 2000). In these methods, activity corresponding to the whole of Band 5 is assayed, so that the influence of TRACP 5a is not negligible. In addition, an immunoassay method has been reported in which TRACP 5b is more specifically assayed (Japanese Patent Application Kohyo No. 2002-510050). It has been reported that this method is an assay method more specific for TRACP 5b activity because in this method, measured values are obtained by activity assay by utilizing the difference in optimum pH between TRACP 5a and TRACP 5b. However, since the antibody used in this method is not specific for TRACP 5b, this method is not always sufficient in specificity for TRACP 5b and hence is desired to be further improved in order to use TRACP 5b as a bone resorption marker.